The expression patterns of minor fibrillar collagens during development in zebrafish

Abstract

Minor fibrillar collagens are recognized as the organizers and nucleators during collagen fibrillogenesis but likely serve additional functions. The minor fibrillar collagens include collagens type V and XI. Mutations of collagens type V and XI can cause Ehlers-Danlos, Stickler's, and Marshall's syndromes in human. We have characterized the spatiotemporal expression patterns of Col11a1, Col11a2, Col5a1 as well as Col5a3 in zebrafish embryos by in situ hybridization. Col5a1 is expressed in developing somites, neural crest, the head mesenchyme, developing cranial cartilage, pharyngeal arches and vertebrae. Col5a3 is detected in the notochord, mesenchyme cells in the eyes and lens. Both Col11a1 and Col11a2 have similar expression patterns, including notochord, otic vesicle, and developing cranial cartilages. Zebrafish may therefore serve as a valuable vertebrate model system for the study of diseases associated with collagens type V and XI mutations.

Figure 1

Sequence and domain structure alignment of minor fibrillar collagens in zebrafish (D. rerio), human (H. sapiens), mouse (M. musculus), rat (R. norvegicus), chimpanzee (P. troglodytes) and chicken (G. gallus). Protein sequence alignment was generated by ClustalW algorithm, zebrafish sequences were predicted based on our sequencing results carried out in this study. A and C) Alignments of peptides encoded by exon 6, 7 and 8 of Col5a1 and Col11a2, respectively; B) Alignment of peptides encoded by exon 6, 7 and 8 of Col5a3; Red arrows indicate the boundaries of exon 6–7 and exon 7–8, respectively; D) Alignments of peptides encoded by exon 6A, 6B, 7 and 8 of Col11a1. Exon assignments are made according to the human sequences, with the exception of Col5a1, exon 4, 5, 6, in which chicken exon sequences were used for the alignment of Col5a1_E6, Col5a1_E7 and Col5a1_E8, respectively; whereas for chimp, exon 7, 8 and 9 correspond to the exons of the VR. (A). Blue letters represent the conserved residue among species. Abbreviations: E6, exon6; E7, exon7; E8, exon8.

Figure 2

Temporal expression and splicing patterns of minor fibrillar collagens. RT-PCR was performed using RNA extracted from wild type embryos at 4, 6, 10, 16, 24, 36, 48, 72hpf. (A). Multiple bands were observed when cDNA was amplified within the region between exons 5 and 9 in the case of Col11a1a on chr24, Col11a2 on chr19, and Col5a3 on chr3. Single amplification products were observed in the case of Col5a1 and Col11a1b on chr2. (B). Splicing patterns are depicted, reflecting the exon usage for those genes undergoing alternative splicing within the variable region. Boxes represent individual exons within the VR, whereas lines in between represent introns. Relative size of all elements depicted reflects relative actual size in basepairs. For Col11a1a on chr24, the predominant spliceform at the earliest stages examined in this study included exons 6A, 7, and 8 within the variable region. Other minor transcripts observed included exons 6A and 6B, or exon 7 alone. For Col11a2, exons 6, 7 or 8 were observed to be either included or excluded in a complex pattern. An additional exon of approximately 1.2kb was observed, replacing exons 6, 7 and 8 in the case of the largest alternatively spliced form. In the case of Col5a3, alternative splicing involved exon 7. (C). Each amplification product was excised and sequenced to confirm identity, intron-exon boundaries, and exon usage in comparison to genomic sequence currently available. Variable region exon sequences in C are color-coded to match the block diagram in B.

Figure 3

Spatial expression patterns of minor fibrillar collagen genes in zebrafish at 16 hpf by whole mount in situ hybridization. The expression patterns of Col5a1 (A, F, K), Col5a3 (B, G), Col11a1b (C, H, L), Col11a1a (D, I, M) and Col11a2 (E, J) are shown in both dorsal (A–E) and lateral (F–J) views. Arrows in D and H indicate expression in the hindbrain. Magnified views of A, H, and D demonstrate Col5a1 expression in neural crest (K), Col11a1b (L) and Col11a1a (M) in hindbrain (arrow in L and lower arrow in M) and otic placode (upper arrow in M).

Figure 4

Spatial expression patterns of minor fibrillar collagen genes in zebrafish at 24 hpf by whole mount in situ hybridization. The expression patterns of Col5a1, Col5a3, Col11a1b, Col11a1a, and Col11a2 are shown in both dorsal (A) and lateral (B) views. Magnified views demonstrate expression in notochord and region of developing somites (C). Magnified view in column 5, row C depicts Col11a2 expression in the developing eye.

Figure 5

Spatial expression patterns of minor fibrillar collagen genes in zebrafish at 48 hpf by whole mount in situ hybridization. The expression patterns of Col5a1, Col5a3, Col11a1b, Col11a1a, and Col11a2 are shown in both dorsal (A) and lateral (B) views. Expression is observed in the perioptic mesoderm, developing craniofacial cartilages, and presumptive retinal pigmented epithelium. Higher magnification views of patterns of expression are shown in Figure 6 for 72 hpf.

Figure 6

Spatial expression patterns of minor fibrillar collagen genes in zebrafish at 72 hpf by whole mount in situ hybridization. The expression patterns of Col5a1, Col5a3, Col11a1b, Col11a1a, and Col11a2 are shown in dorsal (A), lateral (B and C), and ventral (D and E) views. A comparison of overall craniofacial staining is shown in A, C, and D, demonstrating staining in the cranial cartilages including the trabeculae, ceratohyal, ethmoid plate, Meckel’s cartilage, polar cartilage, palatoquadrate, and parachordal cartilage. Expression in the eye is shown in row B. Anterior-most view of developing craniofacial region is shown in row E.